The contractile ring is the transient, actomyosin-based structure responsible for the physical separation of the two daughter cells at the end of mitosis. To ensure the fidelity of sister chromatid transmission into each daughter cell, the formation of the contractile ring must be coordinately regulated both temporally and spatially with the processes of spindle assembly and chromatid separation. However, the molecular mechanisms by which this is accomplished remain elusive, as well as how modifications of these pathways give rise to assymetric cell divisions during development. In an effort to understand the regulation of contractile ring formation during embryogenesis, a detergent-extracted model of the sea urchin zygote cytoskeleton was developed that retains the capacity to reactivate contraction of the contractile ring in vitro, possesses cell cycle regulated kinases able to phosphorylate specific cortical cytoskeletal substrates in vitro and in vivo, and is amenable to structural, biochemical and biophysical analysis. Using these studies as a foundation, this proposal seeks support for a detailed analysis of the role(s) that cell cycle kinases (and their substrates) play in the spatial and temporal regulation of contractile ring formation. The high degree of cell cycle synchrony, as well as the amenability to microinjection and micromanipulation make the sea urchin embryo an attractive model system for the biochemical analysis of cytokinesis. Given the high degree of conservation of cell cycle regulatory mechanisms across phylogenetic lines, it is likely that the results obtained in this study of cytokinesis will be generally applicable. Three outstanding issues regarding the regulation of cytokinesis form the Specific Aims of this proposal. The lines of experimentation described in this proposal will seek to: 1) Define the mechanisms by which cell cycle kinases contribute to the spatial and temporal regulation of contractile ring formation; 2) Characterize the mechanism of myosin activation during cytokinesis; and 3) Identify the molecular determinants that specify the position of the cleavage furrow. It is unlikely that the mechanisms underlying these three aspects of contractile ring regulation are mutually exclusive, and it is expected that the results derived from each line of experimentation will lend insight and direction into the mechanisms addressed in the other two aims. These efforts should lead to a clearer understanding of how the mitotic apparatus, the regulatory machinery of the cell cycle, and cell signaling pathways coordinately act to spatially and temporally regulate cytokinesis.